A deficiency of folic acid leads to a life-threatening megaloblastic anemia. Vitamin B12 deficiency and exposure to the anesthetic gas, nitrous oxide (N20)- causes a functional folate deficiency by trapping of folate coenzymes, needed for DNA and RNA synthesis, as 5-methyltetrahydrofolate. This trapping leads to megaloblastic anemia and to neuropathy. Folates in the liver cell are partitioned between the cytosol and the mitochondria. Recent studies have shown that short-term N20 inactivation of the B12-dependent enzyme, methionine synthase, results in trapping of hepatic folates as 5-methyltetrahydrofolate and greatly decreased concentrations of other reduced folates only in the cytosol. Mitochondrial folate coenzymes were not affected. Therefore, it is quite evident, not only, that folates are compartmentalized in the cell but that nutritional and maybe other factors which affect folate metabolism may do so differently in these cellular compartments. The long-term objective of this proposal is to delineate the subcellular metabolism of folate coenzymes in liver. The effects of long-term N20 exposure and of methionine on cytosolic and mitochondria folate-requiring enzymes will be studied, as will the effects on folate coenzyme and S-adenosylmethionine levels (assayed by high-pressure liquid chromatography). The effects of folic acid deficiency on cytosolic and mitochondrial folate enzyme activity and enzyme protein levels (determined by enzyme-linked immunoassay) and on folate coenzymes will likewise be determined. Mitochondrial transport of folate coenzymes will be studied. Whether mitochondria synthesize S-adenosylmethionine (needed for DNA and RNA methylation) and/or obtain it by transport from the cytosol is not known and will be investigated. The antifolate cancer drug, methotrexate, is being used in long-term treatment of severe psoriasis and rheumatoid arthritis. One of the side effects is hepatic toxicity. The contribution of mitochondrial enzyme inhibition to this toxicity has not been addressed. The distribution of methotrexate between cytosol and mitochondria will be determined. The presence of methotrexate polyglutamate derivatives in these compartments will be assessed, as will the degree of inhibition of folate-requiring enzymes by methotrexate and its polyglutamate derivatives.